This invention relates to the synthesis and use of peptides capable of suppressing the growth of vascular smooth muscle cells. More particularly, this invention relates to the synthesis of novel derivatives of C-type natriuretic peptide (hereunder abbreviated as "CNP"), the novel physiological actions of CNP and its derivatives, and to vascular smooth muscle cell growth suppressing agent that contains one of those peptides as an effective ingredient. The term "CNP analog derivatives" as used herein means the compound recited in appended claim 1, CNP-22, human CNP-53, porcine CNP-53, frog CNP and chick CNP.
Many peptides having natriuretic and hypotensive actions have recently been found in the hearts and brains of various animals. These peptides are collectively referred to as "natriuretic peptides" or "NPs". Many NPs having different chain lengths or similar primary amino acid sequences have heretofore been isolated and identified from living bodies and it has now become clear that all of those NPs are biosynthesized from three different NP precursor proteins (prepro ANP, prepro BNP and prepro CNP).
Therefore, NPs known today can be classified as the following three types in accordance with the route of their biosynthesis: A-type NP (A-type natriuretic peptide or ANP); B-type NP (B-type natriuretic peptide or BNP); and C-type NP (C-type natriuretic peptide or CNP).
Among these NPs, ANP and BNP were isolated from the atrium and the brain, respectively, so ANP has initially been called an atrial natriuretic peptide and BNP, a brain natriuretic peptide (Natsuo, II and Nakazato, H., Endocrinol. Metab. Clin. North Amer., 16, 43, 1987; Sudoh, T et al., Nature, 332, 78, 1988). However, later studies have revealed that ANP is produced not only in the atrium but also in the brain and that similarly, BNP is produced not only in the brain but also in the heart (Ueda, S. et al., Biochem. Biophys. Res. Commun., 149, 1055, 1987; Aburaya, M. et al., Biochem. Biophys. Res. Commun., 165, 872, 1989). It was also verified that both ANP and BNP, when administered in vivo, exhibited comparable and noticeable levels of natriuretic and hypotensive actions. On the basis of those findings, both ANP and BNP are presently considered to work not only as hormones to be secreted from the heart into blood but also as nerve transmitting factors, thereby playing an important role in maintaining the homeostatic balance of body fluid volume and blood pressure.
CNP is a group of peptides that are assignable to a third class of NPs following ANP and BNP and those peptides were isolated very recently, followed by the unravelling of their structures and the mechanism of their biosynthesis.
The first discovered CNPs were CNP-22 composed of 22 amino acid residues and CNP-53 having 31 amino acid residues attached to the N-terminus of CNP-22, and those peptides were both isolated from porcine brain and their structures were unravelled. It was also determined that CNP-22 and CNP-53 were present in nearly equal amounts in the porcine brain (Sudoh, T. et al., Biochem. Biophys. Res. Commun., 168, 863, 1990; Minamino, N. et al., Biochem. Biophys. Res. Commun., 170, 973, 1990). At a later time, porcine CNP genes and cDNA corresponding to CNP-22 and CNP-53 were isolated and their analysis has shown that both CNP-22 and CNP-53 are produced from a common precursor protein (prepro CNP). It was also found that this prepro CNP was clearly different from ANP and BNP precursor proteins (prepro ANP and prepro BNP) (Tawaragi, Y. et al., Biochem. Biophys. Res. Commun., 172, 627, 1990).
The isolation of the porcine CNP gene was followed by the isolation of rat CNP cDNA and human CNP gene and the structures of rat and human CNP precursor proteins have been unravelled. As a result, it has been found that CNP-22 has the same primary amino acid sequence in the three animal species, pig, human and rat, that CNP-53 has the same primary amino acid sequence in pig and rat but has different sequences in human and pig in that amino acid substitution occurs in two positions, and that unlike ANP and BNP, CNP is not produced in the heart but produced specifically in the brain (Kojima, M. et al., FEBS letter, 176, 209, 1990; Tawaragi, Y. et al., Biochem. Biophys. Res. Commun., 175, 645, 1991). As of today, a peptide assignable to CNP has also been isolated and identified from frogs and chicks (Japanese Patent Application Nos. 238294/1990 and 238293/1990).
Thus, it has been verified that CNP occurs not only in mammals but also in birds and amphibians. However, much is left unclear about the physiological role of CNP as NP.
The primary amino acid sequence of CNP is similar to those of ANP and BNP and, when administered in vivo, CNP exhibits natriuretic and hypotensive actions. Therefore, CNP has been held assignable to the NP family. However, compared to ANP and BNP, CNP is considerably weak in natriuretic and hypotensive actions (1/50-1/100) and, further, unlike ANP and BNP, the tissue of CNP production is limited to the brain; thus, CNP stands in a peculiar position in the NP family and it has been speculated that CNP will play other physiological roles in addition to that of maintaining the homeostatic balance of body fluid volume and blood pressure.
The studies conducted so far have shown that the mechanism by which NP exhibits a hypotensive action will probably be as follows: NP binds to an NP receptor present on the surface of a vascular smooth muscle cell, thereby increasing the level of intracellular cGMP (cyclic guanosine monophosphate), which works as an intracellular second messenger of NP to eventually cause the relaxation of blood vessels. As a matter of fact, it has been verified that the level of intracellular cGMP rises when NP is allowed to act on a sample of blood vessel or cultured vascular smooth muscle cells (VSMC).
However, the present inventors recently found that when CNP was allowed to act on VSMC, it unexpectedly increased the level of intracellular cGMP in VSMC several times as much as in the case where ANP or BNP was used (Furuya, M. et al., Biochem. Biophys. Res. Commun., 170, 201, 1990). This suggests that cGMP induced by CNP not only works as a second messenger in vascular relaxation but also has a capacity for functioning as a mediator in the development of other physiological actions. In this regard, Garg et al. have shown that vascular relaxants such as nitroprruside and S-nitroso-N-acetyl penillamine suppress cell growth in a rat VSMC line and that 8-bromo cGMP exhibits a similar action in the same cell line. Garg et al. have concluded that this growth suppressing action is caused by cGMP induced by nitric oxide (NO) radicals (Garg, U. C. et al., J. Clin. Invest., 83, 1774, 1989).
Kariya et al. have reported that ANP enhances the production of intracellular cGMP in cultured vascular smooth muscle cells derived from rabbit aortas, thereby suppressing the growth of those cells (Kariya, K. et al., Atherosclerosis, 80, 143, 1989).
All the reports mentioned above suggest strongly that cGMP works as a mediator in suppressing the growth of cultured vascular smooth muscle cells and they also suggest the possibility that cGMP induced by CNP also suppresses the growth of cultured vascular smooth muscle cells. However, it is not known today whether CNP suppresses the growth of cultured vascular smooth muscle cells.
On the other hand, it is known that all NPs have a cyclic structure composed of 17 amino acid residues that are formed on the basis of intramolecular S--S bonds. Thus, when the structure of NP is divided into three domains including the common cyclic structure (i.e., an exocyclic N-terminal domain, an endocyclic domain, and an exocyclic C-terminal domain), it can be seen that the structure of CNP differs from those of ANP and BNP in the following points (see FIG. 1). First, the primary amino acid sequence of CNP completely differs from that of ANP or BNP in terms of the exocyclic N-terminal domain whereas the endocyclic domain of CNP differs from that of ANP in terms of 5 of the 17 amino acid residues and differs from that of BNP in terms of 4 of the 17 amino acid residues. The structure of the exocyclic C-terminal domain of CNP differs greatly from that of ANP or BNP since it does not have the "tail" structure which occurs in ANP or BNP (in the case of ANP and BNP, 5 or 6 amino acids are attached to the C-terminal side of the cyclic structure of ANP or BNP, respectively, and this structure is named a "tail" structure for the sake of convenience). Obviously, these structural differences between CNP and ANP or BNP contribute to the development of the aforementioned characteristic physiological actions of CNP. However, it has not yet been known as to which domain structure of CNP and which primary amino acid sequence are directly involved in the strong cGMP producing activity of CNP.